Sulfated are central mediators of extracellular traffic and cell-cell communication in humans. The enzymes that install and remove sulfate esters, sulfotransferases and sulfatases, respectively, are now appreciated as major contributors to human health and disease. By contrast, the roles of sulfated sugars and the associated enzymes in bacteria remain relatively unexplored. Mycobacterial pathogens have been declared a global emergency by the World Health Organization, particularly in regard to the deadly synergy of Mycobacterium tuberculosis with AIDS, but also due to the emergence of drug-resistant strains. In Mycobacteria, several sulfated molecules have been identified. These include a sulfated glycolipid, SL- 1 that has been implicated as a virulence factor for M. tuberculosis. Another sulfated carbohydrate, part of a glycopeptidolipid, has been detected in a drug resistant strain of M. aviurn isolated from an AIDS patient. Recently, the complete genome sequences of M. tuberculosis, M. avium, and M. smegmatis have become available, enabling the search for genes that participate in sulfation pathways. We have identified an extensive family of sulfotransferases and sulfatases from the completed genomes of these three Mycobacteria. The enzymes may be critical determinants of Mycobacterial virulence and potential targets for anti-Mycobacterial therapy. Through a collaborative effort, our laboratories (Prof. Carolyn Bertozzi and Prof. Lee Riley, UC Berkeley) have initiated a program aimed at the genetic and biochemical characterization, and small molecule inhibition of the sulfotransferases from M. tuberculosis and M. smegmatis. In addition, we have identified several sulfatases that have considerable similarity to mammalian carbohydrate sulfatases, suggesting a role for these enzymes in host/pathogen interactions. Finally, in order to define the sulfur incorporation pathways of Mycobacteria, we have begun the characterization of enzymes involved in the early stages of cysteine biosynthesis. The aims of this proposal are threefold: (1) to determine the functions of the carbohydrate sulfotransferases in M. tuberculosis and M. smegmatis using genetic, biochemical and chemical approaches; (2) to investigate the involvement of bacterial sulfatases in host/pathogen interactions; and (3) to define the sulfur assimilation pathway of M. tuberculosis.